weight change, *p<0.05 DSS Vehicle + C. rod compared to DSS Vehicle; **p<0.01 DSS Ethanol + C. rod compared to DSS Vehicle by Two-way ANOVA, n=7-8 animals/group. B. Increased colonic shortening with C. rodentium infection after alcohol consumption and DSS- induced colitis. Values are means ± SEM, n=7-8 animals/group. *p<0.05 DSS Ethanol compared to DSS Vehicle; ****p<0.0001 DSS Ethanol + C. rod compared to DSS Vehicle and DSS Vehicle + C. rod ANOVA.
Again, to understand differences in histopathology following infection with C. rod, sections of colon were taken closest to the rectum, stained via H&E, blinded, and scored by a
pathologist. Figure 33A shows gross differences in large intestine morphology after DSS
Ethanol + C. rod treatment compared to all other groups. As in Figure 9B, inflammatory
infiltrate and epithelial damage were assessed and were severely increased in the DSS Ethanol +
C. rod mice compared to that of the DSS Vehicle + C. rod group. The combined histopathology
B.
D SS V eh i cle D SS E th a n ol D SS V eh i cle + C . ro d D SS E th a n ol + C. ro d 0 2 4 6 8 C ol o n Le ng th (c m ) * **** ****scores in DSS Ethanol + C. rod treated mice were significantly increased compared to mice in
Figure 33. Increased Colonic Damage, Inflammatory Infiltrate, and Histopathology Scores with C. rodentium Infection After Alcohol Consumption and DSS-Induced Colitis. A. Increased colonic damage and inflammatory infiltrate with C. rodentium infection after alcohol consumption and DSS-induced colitis. Representative H&E images, n=6-12 animals/group. B. Combined Histopathology Score following blinded histological scoring as described in detail in Methods section above. Values are mean ± SEM, n=6-12 animals/group. **p<0.01 DSS Ethanol + C. rod compared to DSS Vehicle; *p<0.05 DSS Ethanol + C. rod compared to DSS Vehicle +
C. rod and DSS Ethanol by ANOVA.
To further assess colonic barrier breakdown following C. rod infection, we performed an Alcian blue stain, which stains the glycoproteins found in the mucosal layer lining intestinal epithelial cells and in large intestine goblet cells. Eight out of the twelve mice in the DSS Ethanol + C. rod group showed a decrease in the mucus layer along with a decrease in goblet
cells as can be seen in representative images in Figure 34.
D SS V ehic le D SS E th ano l D SS V ehicl e + C . ro d D SS E than o l + C. r o d 0 2 4 6 8 1 0 C o m bi ne d H is topa th ol og y S co re ** * *
B.
Figure 34. Decreased Large Intestine Mucosal Lining and Goblet Cell Number with C.
rodentium Infection After Alcohol Consumption and DSS-Induced Colitis. Representative Alcian blue images, n=6-12 animals/group.
Proper intestinal barrier function is dependent on the formation and integrity of tight
junction protein complexes adhering adjacent intestinal epithelial cells. Figure 35C and 35D
show that C. rod infection results in a significant decrease in expression of both Claudin 8 and Occludin in the large intestine of DSS Ethanol + C. rod mice, respectively. Expression of
Claudin 2 (Figure 35A) and Claudin 4 (Figure 35B) were not significantly altered in the DSS
Figure 35. Decreased Colonic Tight Junction Expression Following C. rodentium Infection with Alcohol Consumption and DSS-Induced Colitis. Values are mean ± SEM, n=6-12 animals/group.
As we did previously, seen in Figure 11, we assessed colonic inflammation under the
hypothesis that C. rod infection would further increase inflammation in mice receiving DSS Ethanol treatment, which could perpetuate increased colonization of C. rod. We found that mice
in the DSS Ethanol + C. rod group had increased levels of IL-18 (Figure 36A) and IL-1β
(Figure 36B) compared to DSS Vehicle + C. rod. Furthermore, the pro-inflammatory cytokine
IL-6 was increased in both the DSS Ethanol + C. rod and DSS Vehicle + C. rod compared to
mice treated with DSS alone (Figure 36C). However, both TNFα and KC were not further
Figure 36. C. rodentium Further Increases Colonic Inflammation After DSS-Induced
Colitis and Ethanol Treatment. Colons were harvested, homogenized, and processed on day 11 for the analysis of inflammatory mediators using respective ELISAs. A. IL-18 *p<0.05 DSS Ethanol + C. rod compared to DSS Vehicle + C. rod and DSS Vehicle by ANOVA, B. IL-1β *p<0.05; **p<0.01; ***p<0.001 DSS Ethanol + C. rod compared to all other groups by
ANOVA, C. IL-6 *p<0.05 DSS Ethanol + C. rod compared to DSS Vehicle and DSS Vehicle +
C. rod compared to DSS Vehicle by ANOVA, TNFα, E. KC by ELISA. Values are mean ± SEM
7-8 animals per group.
Finally, to assess whether mice in the DSS Ethanol group were truly more susceptible to infection, we utilized a bioluminescent strain of C. rod gifted to us by Dr. Sara Jones to image C.
rod colonization of the large intestine following DSS-induced colitis and binge alcohol. The
luxCDABE operon was introduced into the C. rod strain and bioluminescent colonies (lux+)
the DSS Ethanol + C. rod lux+ group have higher levels of photon emission (as seen by increased red and blue areas) in their intestines indicative of increased C. rod colonization compared to DSS Vehicle + C. rod lux+.
Figure 37. Increased C. rodentium Colonization in DSS Ethanol Mice Compared to DSS Vehicle.
Summary
Research has shown that IBD patients and alcoholic patients carry an intestinal bacterial
dysbiosis50,83. A dysbiosis is believed to provide pathogens an opportunity to colonize and
proliferate158 . Indeed, studies have shown IBD patients and alcoholics are at risk for increased
infections159,160. In a retrospective analysis of patients with a documented history of alcohol use
and IBD, our lab found that these patients had increased intestinal infections. With our low inoculate of C. rodentium in our murine model of binge alcohol and DSS-induced colitis, we were able to mimic these patient’s results as DSS Ethanol treated mice had increased
susceptibility and colonization of C. rodentium. We recognize the burden of utilizing the model pathogen, C. rodentium, which itself is used as a model of IBD, on top of our binge alcohol and
DSS-induced colitis model. Yet, our adaptation of using a much lower inoculate, 1 X 105 CFUs
vs. 1 X 1011 CFUs to induce true IBD symptoms, allowed us to shed light on the increased
97
CHAPTER SEVEN DISCUSSION
New Contributions to the Alcohol and Ulcerative Colitis Field
The overarching goal of this work was to expand our understanding of how binge alcohol drinking could potentially worsen GI symptoms of UC and to identify potential treatment
regimens to improve lives of UC patients. While a good amount of work has been and still is dedicated to elucidating a cure for UC, it is imperative that research continues in attempt to not only understand the triggers of flare, but also maintenance therapies to keep patients in remission and out of active UC flare. A report from the U.S. Department of Health and Human Services at the Centers for Disease Control and Prevention stated that the peak age at which patients are
diagnosed with IBD is between 20-401. Interestingly, a study conducted by Naimi et al. found
this is also the age range where an estimated 70% of binge alcohol drinking episodes occur15.
These two independent observations highlight the need to investigate a potential correlation between binge alcohol drinking and UC diagnoses. However, contrasting evidence as to whether drinking alcohol worsens UC symptoms in patient populations made it necessary for our lab to generate a mouse model of UC and binge alcohol drinking.
The results presented here demonstrate that binge alcohol drinking can exacerbate an UC flare period in a mouse model of UC shown by increased weight loss, colonic shortening,
histopathology and clinical scores, inflammation, microbial dysbiosis, failure to release a critical cytokine involved in entrance into remission, IL-22, and susceptibility to infection.
in restoring these exacerbated symptoms in a STAT3-dependent fashion. As a whole, these findings provide a new insight into cellular and molecular mechanisms that may contribute to intestine barrier disruption following an UC flare period in the presence of intoxication, and this may have implication in other forms of autoimmune conditions that acutely affect intestinal physiology.
Generating a Murine Model of UC and Alcohol
One limitation of UC research is that no one murine model exactly replicates UC, as UC pathology is so multi-factorial. Therefore, researchers are restricted to mimicking colitis
symptoms in murine model systems with various chemicals such as DSS, of 2,4,6-
trinitrobenzenesulfonic acid (TNBS), or oxazolone. These chemicals damage the integrity of the large intestine triggering innate and adaptive inflammatory cascades and intestinal dysbiosis, which presents UC-like symptoms. Whether the inflammation or the dysbiosis is the initiating factor in inducing the other is still unknown and a matter of debate amongst UC researchers. Our laboratory choose to utilize DSS in generating our model due to its reproducibility in initiating UC symptoms and ease use. Anywhere from a 1-5% concentration of DSS is used in the field of UC research. Therefore, when generating our model, we first profiled 2, 3, and 4%
concentrations in order to understand not only what works in our hands and laboratory environment, but also to whether DSS produced the expected UC symptoms. The exact mechanism behind which DSS damages the intestinal barrier is still unknown. However, DSS- induced intestinal damage is likely through a combination of disturbance in the metabolism of phospholipids, which are major cellular constituents required for the assembly of biological membranes and loss of tight junction protein ZO-1, which could facilitate in increasing intestinal
permeability. We found that all three concentrations of DSS we utilized, indeed, were able to induce UC-like symptoms in treated mice.
The NIAAA defines binge drinking as a pattern of drinking that brings blood alcohol concentration (BAC) levels to 0.08 g/dL in a single dose. For the past 15 years our lab has had a murine model of alcohol and burn injury, which utilizes a single dose of alcohol at ~3g/kg per mouse. Therefore, we used this alcohol dose when generating our model of UC and binge alcohol. We tested two binge alcohol paradigms, a one-day binge on the last day as seen in Figure 4 and a three-day binge as seen in Figure 6. Our results demonstrated that a 2% DSS concentration followed by a three-day binge alcohol paradigm was the best experimental model to begin to elucidate how alcohol could be perpetuating an UC flare. We were able to show that DSS Ethanol treated mice had exacerbated symptoms of UC as shown by increases in weight loss, colon shortening, more profound histopathology and clinical scores, and increased intestinal inflammation, all of which are standard assessment of UC severity in mouse models. To our knowledge, this is the first time a murine model of UC and alcohol has been developed to allow a better understanding of how drinking alcohol could affect a patient with UC.
However, two questions remain: 1) Whether alcohol can trigger an UC flare? and 2) Whether drinking alcohol predisposes one to a UC diagnosis?
Alcohol Use as a Trigger and/or Predisposition for UC
The results of our work demonstrate alcohol’s ability to worsen an induced flare period (i.e. 5 days of DSS). Once DSS was removed on day 5, DSS Vehicle treated mice showed signs of entrance into remission as seen by less severe histopathology and clinical scores, decreased intestinal inflammation, and increased protein levels of IL-22 compared to DSS Ethanol treated
mice. We recognize the limitation in our alcohol and colitis model that a person experiencing an UC flare might not participate in an alcohol binge.
Therefore, future work will focus on inducing an UC flare with DSS in the drinking water
for 5 days as per our model in Figure 6, but instead of an immediate binge of alcohol, mice will
be allowed to recover for one week to model entrance into UC remission. Then we will implement our three-day binge alcohol paradigm to assess whether a binge of alcohol during a period of UC remission can trigger an UC flare. Preliminary data from our laboratory utilizing this model does indeed show that an alcohol binge can induce UC flare symptoms even during an UC remission period.
To answer the question of whether alcohol could predispose one to UC, we will utilize an IL-10 deficient mouse. IL-10 is an immunoregulatory cytokine essential in the maintenance of intestinal homeostasis. A wide range of immune cells secrete IL-10 such as macrophages, dendritic cells, and T cells, which suppresses effector functions of Th1/Th17 cells as well as NK
cells and macrophages, thereby modulating the cellular immune response162-164. Interestingly, a
genetically engineered IL-10–deficient mouse has been extensively used to dissect IBD etiology.
IL-10 knockout mice were first generated in 1993 by Kühn et al.165. The generation was
performed by use of targeted mutation disrupting the IL-10 gene by replacing a 500 base pair fragment of exon 1 with a termination codon and a neo expression cassette and by introducing a termination codon into exon 3 (Il10tm1Cgn, IL10−/−). IL-10 deficiency produces discontinuous and transmural inflammatory lesions characterized by inflammatory cell infiltrates into the lamina propria and submucosa, epithelial hyperplasia, mucin depletion, crypt abscesses, ulcers,
and thickening of the intestinal wall166,167, all of which are symptoms of human UC. The onset
To answer the question of whether alcohol consumption increases risk of UC diagnosis, we hope to utilize IL-10 knockout mice and employ a binge alcohol paradigm before UC
spontaneously sets in. If a binge of alcohol produced UC symptoms at an earlier time point or in an exacerbated passion, this would provide evidence for alcohol use directly influencing UC risk. Thus, future work will be dedicated in designing a binge alcohol paradigm in IL-10 knockout mice.
Interleukin-22 in UC Remission
One potential pathway that upon its activation could act in the amelioration of DSS- induced colitis is the aryl hydrocarbon receptor (AhR) pathway. Studies have shown that activation of the AhR pathway not only through chemical activation via TCDD (2,3,7,8- tetrachlorodibenzo-pdioxin) but also by specific probiotics is able to inhibit DSS-induced
colitis132,169 . Additionally, cytokines known to be induced upon AhR activation, specifically IL-
22, are known to be upregulated during remission periods of UC98,143 .
IL-22 remains one of the most intriguing cytokines due to its ability to elicit completely different responses based on the microenvironment. In the context of the intestines, the presence of IL-22 appears to be beneficial under most circumstances including inflammatory bowel
disease, graft-versus-host disease, and many types of bacterial infection102,153,170-173.
Interestingly, certain bacterial infections, such as in mouse models using T. gondii, cause IL-22
to be pro-inflammatory174,175. While the reasons behind these differential responses of IL-22
remain unknown, it illuminates the importance of understanding the role of IL-22 under different conditions. In addition, current clinical trials using Fc-fusion IL-22 administration for treatment of patients with graft-versus-host disease have shown promising preliminary results, indicating
The increased weight loss, colonic shortening, more profound histopathology and clinical scores, and increased intestinal inflammation outlined in Chapter 3 clearly demonstrate alcohol’s role in increasing symptoms of UC flare. Combined with the knowledge that IL-22 is a critical cytokine for entrance into UC remission, we examined whether alcohol was affecting IL-22 levels and thus perpetuating UC flare. Our results show that mice allowed to recover for 3 days (DSS Vehicle group) experienced increased levels of IL-22 in large intestine homogenates. In mouse models of colitis, the innate immune response in the colon includes recruited
macrophages and neutrophils, which appear to have both pro-inflammatory and anti-
inflammatory roles in colitis177. Specifically, aberrant control of these infiltrating neutrophils
can result in tissue damage in the colon caused by abundant reactive oxygen species178-180.
However, recent studies demonstrated that neutrophils can play a protective role in the host
response in acute models of colitis by producing IL-22181,182. Hence, we examined whether the
spike in IL-22 levels in DSS Vehicle mice was due to infiltrating neutrophils, and conversely whether the diminished IL-22 response in DSS Ethanol mice was a consequence of abnormal control of IL-22+ producing neutrophils. We found that both large intestines of DSS Vehicle and DSS Ethanol mice had increased percentage of neutrophils. In contrast to Zindl et al.’s results, these isolated neutrophils were not producing IL-22 in our model of DSS-induced colitis and alcohol.
However, examination IL-22+ γδ T cells revealed that DSS Vehicle treated mice had a significantly increased percentage of IL-22+ γδ T cells, but this response was abolished in DSS Ethanol treated mice. This balance between decreased percentages of IL-22+ neutrophils and simultaneous decrease in percentages of IL-22+ γδ T cell in DSS Ethanol mice gives evidence to how alcohol could be damaging the critical response IL-22 needed to restore the integrity of the
intestine following mucosal injury and enter into UC remission. Therefore, increasing levels of large intestine IL-22 in the context of binge alcohol consumption and UC could act a potential therapeutic target to improve lives of UC patients.
In the intestine IL-22 can stimulate proliferation, mucous protection, and AMP secretion,
which could drive entrance into the remission period from a UC flare99,104,109,111,182,183. The
present study provides a mechanistic role for IL-22 mediated protection against alcohol induced UC flare through STAT3. A recent report supporting this mechanism from Hanash et al. showed that IL-22 specifically signals through STAT3 in stem cells within the crypts of both small and
large intestines to promote barrier regeneration in a murine model of graft versus host disease184.
While IL-22 has been shown in other models to signal through other STATs or MAPK/ERK
pathways185-188, we found epithelial cell STAT3 to be necessary for IL-22 mediated protection
against alcohol-induced exacerbation of DSS-induced colitis.
Although this is an exciting finding for the field of alcohol and colitis research,
translating this treatment of an exogenous protein into UC patients could possible have systemic side-effects. Therefore, we chose to examine probiotics as a potential therapeutic option has they are readily available without a prescription.
Probiotic Intervention as a Potential Therapeutic Option
Interestingly, we also found that treatment with the probiotic, Lactobacillus delbrueckii, attenuated the increased severity of UC symptoms we observe after DSS-induced colitis and alcohol. Lacto treatment was able to upregulate activated colonic STAT3 following colitis and alcohol back to that of DSS Vehicle treated mice providing mechanistic evidence for how
We acknowledge that this work does not currently have any functional read outs of the benefits of Lacto treatment. As activated STAT3 is known to increase intestinal epithelial cell proliferation, increase AMPs, and increase immunomodulatory cytokines such as IL-10, we expect Lacto treated mice to will also have increases in these downstream targets of activated STAT3. Additionally, we expect Lacto treatment to increase percentages of IL-22+ lamina propria immune cells in DSS Ethanol treated mice. Our findings in Chapter 5 not only gives evidence to γδ Tcells as the potential source of IL-22 in DSS Vehicle treated mice, which models entrance into UC remission, but also points to an inability of γδ T cells to mount the proper IL-22 response in the presence of alcohol. Finally, we expect FACS analysis of large intestine lamina